The QIAamp cador Pathogen Mini Kit simplifies the isolation of viral RNA and DNA and bacterial DNA from a whole range of animal samples, including undiluted whole blood, serum, swabs, and tissue. Using a single, rapid spin-column procedure, contaminants and inhibitors are removed to yield pathogen nucleic acids that are ready for use in downstream applications, such as real-time PCR and RT-PCR. The procedure can be fully automated on the QIAcube.

For laboratory use. Not for use in veterinary diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a veterinary disease.

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High repeatability of nucleic acid isolation.|Successful isolation of bacterial DNA.|Comparison of manual and automated processing.|

Whole blood samples from various animals were spiked with RNA virus particles. The samples were processed in triplicate on 3 separate days. The data from the RT-PCR show high repeatability with error bars representing ±1 S.D.|Various animal samples were spiked with Gram-negative or Gram-positive bacteria. If necessary, the appropriate pretreatments were applied (B1 for bovine blood, B2 for swab medium, and an adapted version of B2 for bovine feces). The data from the PCR shows that the isolation of bacterial DNA was successful in all cases. The error bars represent ±1 S.D. from 3 QIAcube runs.|Bovine serum was spiked with RNA virus particles and the isolation protocol was run manually and on the QIAcube. The RT-PCR amplification data for the purification duplicates show high and identical isolation efficiency and linearity. Gray lines: manual isolation. Blue lines: automated isolation. Red lines: internal control.|

Data show that high and identical purification efficiency can be achieved with both the manual and automated protocols for viral RNA isolation (see the figure "Comparison of manual and automated processing."). Bovine serum was spiked with virus particles from cell culture. The sample was serially diluted 1:10 with bovine serum and processed in duplicate using the QIAamp cador Pathogen Mini Kit either manually or using the automated protocol on the QIAcube. Viral RNA was detected using the QuantiFast Pathogen RT-PCR +IC Kit with target-specific primers and probe.

The robustness of the protocol is demonstrated by the high intra- and inter-assay repeatability (see the figure "High repeatability of nucleic acid isolation."). Whole blood samples from different animals were spiked with virus particles from cell culture and then frozen at -20°C. Three replicates for each sample were processed on three different days with the QIAamp cador Pathogen Mini Kit. The viral RNA was amplified with the QuantiFast Pathogen RT-PCR +IC Kit using target-specific primers and probe.

The kit also successfully isolates bacterial DNA from animal samples, as shown in the figure "Successful isolation of bacterial DNA". Different animal samples were spiked with Gram-negative or Gram-positive bacteria and pretreated as necessary (see the table "Pretreatments for the QIAamp cador Pathogen Mini Kit protocol"), and then the automated isolation protocol was run on the QIAcube. The bacterial DNA was successfully purified. PCR setup with parallel preparation of reaction mixes for both bacterial targets was performed using the QIAgility, and the bacterial DNA was amplified with the QuantiFast Pathogen +IC Kit with target-specific primers and probes.

Principle

The QIAamp cador Pathogen Mini Kit combines the features of several sample preparation kits based on spin-column technology to permit the purification of viral RNA and DNA and bacterial DNA from animal fluid and tissue samples. The spin-column procedure uses optimized buffers and enzymes to lyse samples and stabilize the pathogen nucleic acids. This RNA and/or DNA binds to the QIAamp silica membrane, while contaminants pass through. Wash buffers are used to completely remove PCR inhibitors, such as divalent cations and proteins, and the pure pathogen nucleic acids are then eluted in Buffer AVE.

QIAamp cador Pathogen Kit technology yields pathogen RNA and DNA from animal samples that is ready for use in downstream applications such as real-time PCR and RT-PCR.

In contrast to other kits based on silica membranes, up to 200 µl of undiluted animal whole blood samples can be processed with the QIAamp cador Pathogen Mini Kit without clogging the filter. See the table "Key features of the QIAamp cador Pathogen Mini Kit".

Most common fluid samples can directly be processed using the main protocol, which involves the lysis, binding, wash, and elution steps described in the "Principle" section.

Tissue samples and samples containing difficult-to-lyse bacteria require the appropriate pretreatment (see the table "Pretreatments for the QIAamp cador Pathogen Mini Kit protocol"). After pretreatment, the samples undergo the same procedure as for fluid samples (see the flowchart "Processing of various sample types and targets"), enabling parallel processing either manually or in an automated procedure on the QIAcube.

Pretreatments for the QIAamp cador Pathogen Mini Kit protocol

Name

Application

Pretreatment B1

For difficult-to-lyse bacteria* in whole blood or pretreated tissue

Pretreatment B1

For difficult-to-lyse bacteria* in whole blood or pretreated tissue

Pretreatment B2

For difficult-to-lyse bacteria* in cell-free fluids

Pretreatment B3

For easy-to-lyse bacteria in high-volume cell-free fluids

Pretreatment T1

Mechanical disruption of tissue for extraction of viral RNA and DNA

Pretreatment T2

Enzymatic digestion of tissue for extraction of viral and bacterial DNA

Pretreatment T3

Rapid partial disruption of tissue for extraction of viral RNA and DNA and bacterial DNA

Pretreatment T4

Organic extraction for extraction of viral RNA and DNA and DNA from easy-to-lyse bacteria* from difficult tissue

* Gram-positive bacteria are difficult to lyse due to their rigid cell wall. However, some Gram-negative bacteria are also difficult to lyse, and benefit from pretreatment B1 or B2.